Antibodies which recognize and bind human villin

ABSTRACT

Antibodies, both polyclonal and monoclonal, recognize and bind human villin. Hybridomas, producing monoclonal antibodies which bind human villin, are also provided.

This application is a continuation of application Ser. No. 07/989,696filed Dec. 14, 1992, now abandoned, which is a continuation of Ser. No.07/662,992 filed, Feb. 28, 1991, now U.S. Pat. No. 5,188,967, which is adivisional of Ser. No. 07/529,676, filed May 30, 1990, now abandoned,which is a continuation of Ser. No. 07/287,658 filed Dec. 21, 1988, nowabandoned, which is a continuation of Ser. No. 07/006,717, filed asPCT/FR86/00150 Apr. 30, 1986, now abandoned.

The invention is related to agents for the in vitro diagnosis of theexistence in man of proliferations of malignant cells derived fromtumour cells of the digestive tract, more particularly of theadenocarcinoma type. The agents refer to both in vitro diagnosticprocedures and the materials used to implement them, more especiallyfragments of DNA to be used as probes or even antidbodies, preferablymonoclonal, directed against human villin.

The invention also covers various applications of such monoclonalantibodies, for example, the purification of human villin from cellextracts containing it. In this sense, the invention also relates topurified villin itself.

It has already been suggested that the detection of structural proteinsas specific markers be employed as a means to identify certain types ofcells in higher eucaryotes and for monitoring the various stages oftheir development, particularly in the course of differentiation.Examples of known markers which may be mentioned are the proteins offilaments, such as vimentin, keratins or proteins of neurofilamentswhich have enabled certain types of cells to be identified and theirdevelopment to be studied from the stage of embryogenesis to the finalstage of cellular differentiation, as well as in relation to theirenvironment (E. LAZARIDES, Nature, 283, 249 (1980) and R. Moll et al.,Cell 31, 11 (1982).

The keratins are often considered to be particularly effective markerssince they allow a distinction to be made between epithelial andnon-epithelial cells. This being said, it has been found that this typeof distinction does not always allow the origin of the epithelial cellsin question to be unambigously established.

However, the need to establish the origin of certain types of cells withcertainty assumes particular importance in a large number of cases as,for instance, when examination of a biological specimen has providedevidence of tumour cell proliferation, it is necessary to identify theprimary tumour cells responsible for the proliferation or it isnecessary to detect the existence of metastases which might originatefrom a cancer of the digestive region or to verify the efficacy of achemotherapeutic or other type of treatment of metastases. It is wellknown that the efficacy of an anticancer treatment is dependent on aprecise identification of the tumour cells which are the primary causeof the disease.

These comments apply with particular force to the detection, preferablyearly, of proliferations of tumour cells of digestive (responsible for aconsiderable proportion of cancers observed in the clinic) or renalorigin, or of cells derived from them, more particularly to theidentification of characteristic markers of these cells.

Thus, the invention relates more particularly to the detection of tumourcells which have preserved some of the essential phenotypes of theepithelial cells usually present in the mucous membranes of thedigestive apparatus, more especially in the intestinal muccsa, and theprincipal differentiated types of which are composed of absorbant cells,or enterocysts.

More particularly still the invention relates to specific markerscapable of being detected, whether or not they are carried by tumourcells.

In particular, an aim of the invention is to make it possible to detectsuch markers, even when they have become detached from the said tumourcells, in particular as a result of cell necrosis, and released, forexample, in to the general circulation.

The marker involved in the context of the present invention is made upof all or part of the villin molecule. The latter is a protein with amolecular weight of about 95,000 daltons and is normally present in thevilli of the digestive mucosa, more especially in intestinal villi.Villin is able to bind to actin in the presence of calcium ions.

Although its amino acid sequence may differ slightly from one species toanother, the functional characteristics are maintained intact. Thetechniques of purification presently available have led to chicken andporcine villins being obtained in an essentially pure state. On theother hand, it has not been possible to obtain purified human villin bythese techniques.

One of the best known of the villins is that obtained from chicken. Itconsists of a sequence of 854 amino acids. Proteolysis by trypsin andV-8 protease leads to cleavage at specific sites with the production of44 Kd, 51 Kd and 10 Kd fragments, the relative positions of which in thevillin molecule have been established by Paul MADSUDAIRA, MRC,Laboratory of Molecular Biology, Cambridge, U.K. and John GLENNEY etal., J. B. C., 1981, 256, 8156-8161, to be the following: ##STR1##

The amino acid sequence of the COOH terminal part of the molecule(779-854) or head piece (or HP peptide) of chicken villin is as follows:##STR2##

As a general rule, villin is found more especially in brush borderswhich are observed on the surface of enterocysts, particularly afterthey have attained their final stage of differentiation, at the level ofthe walls of the intestinal lumen. The villi are organelles assembled atthe final stage of differentiation of the enterocyst. Villin islocalised particularly in the bundles of axial microfilaments of suchvilli. It contributes to the structure of their cytoskeleton. Onexamining frozen sections by immunofluorescence, it has been observedthat villin is present at the apical pole of elongated cells formingcolumns at the surface of the internal wall of the intestine, and alsoclose to cells of the proximal tubules of the kidney. In both casesthese regions are contiguous with the brush border of the cellsconcerned. However, villin has not been detected in various other typesof villi of various other epithelial cells when the villi were notprovided with a well organised brush border (A. BRETSCHER et al., Exp.Cell Res., 135, 213 (1981) or the article by H. REGGIO et al., publishedin the monograph entitled "Membranes in Growth and Development" by A.LISS, New York (1982) pp. 89-105).

Futhermore, it has been observed that villin is present in enterocystsfrom an early stage of their development, well before the organisationof the brush border.

The invention is the consequence of a double discovery. Villin canalmost always be detected throughout the course of tumorogenesis of thetissues of the digestive tract, and particularly in cancers of thecolon, one of the most widespread forms of cancer, and also in certaintypes of renal cancer, whereas villin has never been detected in primarytumour cells derived from cancers localised in the tissues of otherorgans (liver, ovaries, lungs etc.).

Moreover, villin can be detected in vivo at all stages of thedevelopment of tumour cells in the digestive region, at both an earlyand later stages of tumorogenesis, irrespective of the state ofdifferentiation of the cells concerned.

The diagnostic procedure according to the invention, the aim of which,essentially, is the in vitro detection in a biological specimen oftumour cells originating from the digestive region and/or derivatives ofthem, is characterised in that this biological specimen is placed incontact with reagents exhibiting a specific affinity for villin orrecognizing the gene coding for this protein.

The diagnostic procedure according to the invention is applicable to anybiological specimen likely to contain tumour cells of digestive originor tumour cells formed elsewhere in the organism under the influence ofprimary cells of digestive or renal origin, or even to any biologicalspecimen likely to contain degradation products of these various tumourcells, including the villin marker, produced by necrosis of the cells.This biological specimen may be made up of all types of specimens:fragments of tissue or of solid tumours obtained, for instance, atbiopsy, or liquid specimens, particularly whole blood or serum, likelyto serve as a vehicle for malignant cells or fragments derived fromthem.

According to one embodiment of the invention, the reagent used in thediagnostic procedure of the invention is composed of DNA, the sequenceof which contains a region coding for an amino acid sequence of humanvillin, more particularly the COOH-terminal part.

According to another embodiment of the invention, the techniquesemployed in the diagnostic procedure makes use of immunological methods,the reagent exhibiting a specific affinity for villin being composed ofantibodies capable of recognizing villin.

Once bound to villin or to tissues containing it, these antibodies, inturn, are labelled by any known technique or are recognizable bylabelled immunoglobulins or other labelled proteins (for example, the Aprotein of Staphylococcus aureus).

Particularly appropriate techniques are those which have been employedin the assays described later and which have led to observations set outabove.

The aim of the invention is also to provide new specific agents todetect villin in the villi of enterocysts of similar cells (it isunderstood that the use of these new agents is not necessarily limitedto the employment of the diagnostic procedure according to theinvention).

According to one aspect of the invention, these agents are composed ofthe DNA corresponding to the complete mRNA sequence of human villin orof a DNA fragment, the nucleotide sequence of which contains a regioncoding for a sequence of amino acids found in human villin, capable ofhybridising specifically with a nucleotide sequence coding for villin.

The invention relates more particularly to the use in the aboveprocedure of a DNA corresponding to the complete mRNA of human villin orof a fragment of cDNA characterised by the fact that it contains atleast part of the nucleotide sequence coding for the corresponding aminoacid sequence of the COOH-terminal of human villin. The nucleotidesequence and the amino acid sequence for which it codes are given below:

    __________________________________________________________________________    LYS TRP SER ASN THR LYS SER TYR GLU ASP LEU LYS ALA GLU                       AAG TGG AGT AAC ACC AAA TCC TAT GAG GAC CTG AAG GCG GAG                       SER GLY ASN SER ARG ASP TRP SER GLN ILE THR ALA GLU VAL                       TCT GGC AAC TCT AGG GAC TGG AGC CAG ATC ACT GCT GAG GTC                       THR SER PRO LYS VAL ASP VAL PHE ASN ALA ASN SER ASN LEU                       ACA AGC CCC AAA GTG GAC GTG TTC AAT GCT AAC AGC AAC CTC                       SER SER GLY PRO LEU PRO ILE PHE PRO LEU GLU GLN LEU VAL                       AGT TCT GGG CCT CTG CCC ATC TTC CCC CTG GAG CAG CTA GTG                       ASN LYS PRO VAL GLU GLU LEU PRO GLU GLY VAL ASP PRO SER                       AAC AAG CCT GTA GAG GAG CTC CCC GAG GGT GTG GAC CCC AGC                       ARG LYS GLU GLU HIS LEU SER ILE GLU ASP PHE THR GLN ALA                       AGG AAG GAG GAA CAC CTG TCC ATT GAA GAT TTC ACT CAG GCC                       PHE GLY MET THR PRO ALA ALA PHE SER ALA LEU PRO ARG TRP                       TIT GGG ATG ACT CCA GCT GCC TTC TCT GCT CTG CCT CGA TGG                       LYS GLN GLN ASN LEU LYS LYS GLU LYS GLY LEU PHE *(SEQ. ID. NO. 2)             AAG CAA CAA AAC CTC AAG AAA GAA AAA GGA CTA TTT TGA GAAG                      AGTAGCTGTGGTTGTAAAGCAGTACCCTACCCTGATTGTAGGGTCTCATTTTCTCA                      CCGATATTAGTCCTACACCAATTGAAGTGAAATTTTGCAGATGTGCCTATGAGCAC                      AAACTTCTGTGGCAAATGCCAGTTTTGTTTAATAAATGTACCTATTCCTTCAGAAA                      GATGATACCCCAAAAAAAAAAAA (SEQ. ID. NO. 1)                                      __________________________________________________________________________

The nucleotide sequence concerned is used as a probe for the detectionof mRNAs or DNAs coding for human villin.

A probe appropriate for this type of detection is advantageouslylabelled with a radioactive element or any other grouping permitting itto be recognised after hybridisation with the preparation containing themRNA or DNA to be studied.

Following standard techniques, these probes are placed in contact with abiological specimen containing the cells under investigation or directlywith their nucleic acids under conditions which allow hybridisation tooccur between the nucleotide sequence of the probe any complementarysequence contained in the sample tested.

The reproducibility of detection was tested using a probe containing aninsert of 200 base pairs.

These nucleotide probes, which constitute new products forming part ofthe invention, are also used, according to another aspect, to study theexpression of villin mRNAs at each stage of differentiation of theenterocysts and/or cells expressing villin, and in the various stages ofmaturation of renal or intestinal cells.

The probes thus prepared make it possible to determine the number,organisation and structure of the gene(s) coding for villin startingfrom a human gene bank.

In conformity with the invention, the nucleotide sequence coding for theterminal portion of human villin is isolated by means of the followingprocedure:

total mRNA (messenger RNAs) is prepared according to known techniquesfrom a cell line expressing villin

a cDNA (complementary DNA) bank is constructed;

these cDNAs are inserted into a vector capable of expressing the proteincoded by the insert;

by means of the recombinant vectors obtained a bacterial strain istransformed, then conditions permitting the expression of the desiredprotein in the bacteria are established;

the recombinant clones containing the clone specific for villin isselected by means of antibodies recognizing villin.

The step leading to the setting up of the cDNA bank is carried out bymeans of a vector capable of expressing the protein encoded in theinserted cDNA.

Plasmids of the type pEX are particularly advantageous cloning vectors,especially on account of their high yield of expression. Such plasmidshave been described by Stanley and Luzio in EMBO Journal, 1984, 3,1429-1434. They describe plasmids containing the cro-lacZ gene fusion,the expression of which is under the control of the promoter P_(R) ofthe bacteriophage lambda. Its expression is temperature-inducible.

A polynucleotide containing several uniques restriction sites isinserted in each of the three possible reading frames, as well as thesignals for termination of transcription and translation. The cDNAsinserted at the unique restriction sites are expressed with greatefficiency as protein in the form of corresponding hybrid cro-βgalpeptide. The poor solubility of this protein hybrid advantageouslyreduces the risks of proteolysis in the bacterium and facilitates itsimmunological detection by specific antibodies.

In order to obtain expression of the corresponding polypeptide in thebacteria a protocol for the sequential addition of different linkers tothe two ends of the cDNA according to the technique of Helfmann et al.in P.N.A.S. USA, 1983, 80, 31-35 is employed in order to ensure that thecDNA is inserted in the correct orientation with respect to the cro-lacZgene. The linkers used correspond to the BamHI and SalI cleavage sites.

These recombinant plasmids are used to transform the bacterial strainemployed, according to standard techniques, so that the protein of thedesired sequence can be expressed.

Strains of E. coli bacteria are particularly preferred, most especiallythe strain E. coli pop 2135 which contains the allele cIts857 of the crorepressor.

A bacterial strain is thus obtained containing a cDNA bank correspondingto the mRNA of the cells expressing a high level of villin, containingabout 30,000 recombinant clones.

The expression of the protein hybrid is temperature-inducible. Thus, aworking temperature is chosen at which the cro gene repressor isinactive. For this purpose, it suffices to incubate the strains for 2hours at a temperature higher than room temperature, in particular atabout 40°-42° C.

The clones recovered after lysis of the bacteria and renaturation of theproteins according to standard techniques are selected by immunologicalscreening.

In a preferred manner, screening of the cDNA bank is first carried outby at least one type of polyclonal anti-villin antibody. The clonesreacting specifically with the polyclonal antibody of antibodies arerescreened by means of at least one type of monoclonal antibody directedagainst epitopes of the COOH-terminus.

Satisfactory screening is achieved by means of a polyclonal anti-villinantibody such as a polyclonal antibody directed against intact porcinevillin, followed by one or more monoclonal antibodies recognizingepitopes located in the COOH-terminal region of the molecule.

Screening with a polyclonal antibody is advantageously followed by asecondary screening with another polyclonal antibody, in particular apolyclonal antibody directed against the COOH-terminal fragment ofchicken villin.

The invention relates more particularly to a clone carrying a cDNAcorresponding to human villin, characterised in that it reactsspecifically with a polyclonal antibody directed against intact villin,with a polyclonal antibody directed against the COOH-terminal peptide ofchicken villin and with two monoclonal antibodies directed againstCOOH-terminal epitopes.

The invention also relates to a clone bearing a cDNA corresponding tovillin comprising an insert with a poly A sequence and apoly-adenylation site.

According to another aspect of the invention, the agents implemented forthe detection of villin consist of monoclonal antibodies.

This aspect of the invention is based on the fact that villins fromseveral species possess common epitopes, accessible to monoclonalantibodies recognizing specifically not only the particular villin usedto immunise the animal, the spleen cells of which were fused withappropriate myeloma cells for the production of hybridomas secreting thedesired monoclonal antibodies, but also recognizing specifically villinsof other species, including human villin.

A preferred monoclonal antibody according to the invention is thuscharacterised by the following properties:

It recognises purified porcine villin (Western Blotting).

It recognises chicken villin (Western Blotting).

It recognises villin extracted from a cell line derived from anadenocarcinoma of human colon: HT29 (Western Blotting).

It recognises rat villin in immunocytochemistry (cryostat sections ofintestinal mucosa of the rat fixed with formaldehyde).

A preferred monoclonal antibody among those satisfying the precedingdefinition also recognises an epitope contained in the "HP peptide" ofchicken villin, recognition being manifest by both the isolated peptideand by the 51 Kd peptide still containing the HP peptide; this antibodydoes not recognise either the 44 Kd peptide nor the 44 Kd portion ofchicken villin.

Moreover, the preferred antibody of the invention recognises the HPprotein both in the denatured state, in particular after treatment withsodium dodecyl sulfate (SDS), and in epithelial cells containing villin.This result demonstrates that the epitope carried by the HP peptide isnot masked by other proteins in the cellular environment.

The invention obviously also relates to the hybridomas secreting thesemonoclonal antibodies. An advantageous technique for the preparation ofthese hybridomas, in particular by fusion of spleen cells of miceimmunised against purified porcine villin, on the one hand, with cellsof an appropriate myeloma, will be described later. The preferredhybridoma thus obtained (strain BD-ID2C3) has been deposited in theCollection Nationale des Cultures de Micro-organismes (CNCM) at thePasteur Institute, Paris under No. I-440, on Apr. 29th 1985.

According to an advantageous aspect of the invention, the immunogen usedto prepare the antibodies can be expressed in the bacterium in largequantities. This immunogen is represented by the peptide encoded in thenucleotide sequence defined above corresponding to the DNA of humanvillin or a fragment of it.

Following standard techniques, the peptide is injected into animals, theantisera are recovered and, if required, the antibodies are isolatedfrom the antisera by, for example, affinity chromatography. Theproduction of antibodies according to that variant assumes a greatersignificance in that the HP region encoded in the cDNA fragment definedabove is both the most potent immunogen and the most specific region ofvillin.

It will be noted that the protein encoded in the nucleotide sequence ofthe villin complementary DNA or a fragment of this DNA also represent asource of antigens enabling competitive radio-immunoassays to be carriedout (either by the direct ELISA method or by the competitive ELISAmethod).

However, it is obvious that any other immunogen containing the sameimmunogenic sequence can be used; for example, the HP peptide itself,grafted on to a carrier molecule such as bovine serum albumin beforehandif necessary, in order to augment its immunogenic properties.

It will be clear to the specialist that, having on hand the preferredmonoclonal antibody according to the invention, it is even possible todefine the characteristic epitopic region of the HP peptide. In order toidentify it more precisely, it would be necessary to use a procedurecomprising the following steps:

synthesis of a polynucleotide coding for the HP peptide (or for the partof the HP peptide which may be reasonably considered to contain thedesired epitope),

linearisation of the plasmid defined above containing the nucleotidesequence coding for the COOH-terminus of villin, and at a restrictionsite located outside of this sequence,

trimming of the linearised plasmid in a controlled manner with anexonuclease, such as 3al 31,

religation of the plasmid by means of a DNA ligase,

transformation of an appropriate micro-organism, transformable by thecorresponding plasmid and capable of expressing the insert contained inthe latter,

placing of the products expressed by this micro-organism in contactagain with the antibody under consideration.

the cycle of operations just described being repeated until theimmunogenic peptide can no longer be detected in the products expressedin the transformed micro-organism by the religated plasmid.

At the end of each of the cycles of the procedure described above, inparticular by sequencing the ends of the plasmid before and after thetrimming operation which leads to the loss of the religated plasmid'scapacity of recognition, it is possible to locate the epitope at thelevel of the peptide encoded by the nucleotide sequence eliminatedduring the last trimming operation. That is to say that the fact thatthe specialist has at his disposal the monoclonal antibody indicatedabove is equivalent to his possessing the chemically defined peptidesequence and containing that epitope.

The invention also relates to human villin, practically pure from abiological standpoint, reacting with the preferred monoclonal antibodydefined above and giving essentially only one band on polyacrylamide gelelectrophoresis, in particular SDS-PAGE.

In fact, human villin can be extracted from a lysate of humanenterocysts, in particular as obtained by treatment of the latter withan aqueous solution containing an appropriate detergent, and purified bymeans of the above-mentioned monoclonal antibody. A purificationprocedure of this kind makes use of a monoclonal antibody advantageouslyimmobilised on a solid support, preferably suited to the operations ofaffinity chromatography. For example, the monoclonal antibody isattached to a three-dimensionally cross-linked agarose network, marketedunder the name of SEPHAROSE by the Swedish company PHARMACIA AG, by thecyanogen bromide method.

Thus, the invention relates more particularly to a separation procedurefor human villin characterised by the operations consisting of passing asolution of it over an affinity column bearing the above-mentionedmonoclonal antibody in order to selectively bind human villin, and thenrecover it by dissociation of the antigen-antibody complex, either by aglycine-based acid buffer at pH 2-4 or by a methylamine-based basicbuffer at pH 11, then of dialysis against an ammonium acetate buffer.

It is obvious that the invention also relates to lower molecular weightpolypeptides consisting of fragments of human villin. It will be clearto the specialist that the fragments may be obtained by cleavage ofhuman villin by proteases at specific sites. As examples of suchenzymes, mention will be made, in the first place, of trypsin and the V8protease of Staphylococcus aureus, alpha chymotrypsin, mousesubmaxillary gland protease, marketed by the Boehringer company, thecollagenase from Vibrio alginolyticus chemovar iophagus, whichspecifically recognises the peptides Gly-Pro and Gly-Ala, etc.

It goes without saying that the hybridomas and monoclonal antibodies,species-specific or not, which may be produced starting from humanvillin or its fragments also form part of the invention.

Other characteristics and advantages of the invention will appear in thedescription which follows of special conditions, indicated as examples,of detection of human villin, of the production and utilisation inassays of hybridomas and monoclonal antibodies.

A procedure for obtaining a clone bearing a portion of cDNA of humanvillin is also reported.

EXAMPLE 1 Detection of Human Villin

The expression of villin can be studied at different stages of theproliferation of normal and tumour cells originating in the digestive orrenal regions, on the one hand, and in other regions of the organism, onthe other. The tissues of the organs concerned are frozen, fixed andprepared under conditions permitting the application of the techniquedescribed by BROWN and FARQUHAR, Cell 36, 295 (1984) to give cryostatsections.

Sections 5 μm thick were obtained at -25° C. and deposited on glassslides coated with polylysine. After being immersed in a buffer solutionconsisting of a phosphate salt solution containing 0.2% of gelatine(PBS-gelatine), the sections were treated with a solution containing 50μl of rabbit anti-villin antiserum diluted 1/800 in PBS-gelatine inorder to detect the presence of villin, then incubated for 25 minutes atroom temperature in a humid chamber.

The sections were then washed three times for ten minutes inPBS-gelatine.

Rabbit anti-IgG labelled with rhodamine (obtained from the Dutch companyNordic Immunological Laboratories) were then added and the sections wereincubated in the presence of these anti-IgG at room temperature for 25minutes. The sections were then washed thoroughly in PBS-gelatine andmounted for examination under a fluorescence microscope (ZEISSphotomicroscope) fitted with a PLANNEOFLUAR oil immersion lens and aseries of appropriate filters.

The control tissues or tumour tissues from other sources were treated inthe same manner.

The following observations were made:

When the tissues were derived from adenocarcinomas of the colon, 11 ofthe 12 specimens gave a positive result for the presence of villin.Human tumours originating in other organs such as lymphomas, inparticular lymphomas of mesenteric nodules and various types ofcarcinomas originating in other organs and giving rise or not tometastases of the pancreas and oesophagus, did not express this protein.

Generally speaking, a close correlation was observed between the primaryintestinal origin of the tumours examined and the expression of villin.Numerous assays were carried out on tumours of obviously other thanintestinal origin and the expression of the protein was revealed to benegative in practically all cases.

It is to be noted that villin was always observed in tissues ofintestinal origin, whatever the degree of proliferation of the cells inquestion.

The preceding assays were carried out with polyclonal antibodies. Muchmore clear-cut results were obtained with the preferred monoclonalantibody which was left in the custody of the CNCM.

The conditions under which the hybridomas secreting these monoclonalantibodies were isolated will be described later in examples. Apreferred mode for the implementation of the conditions under which thepresence of villin can be diagnosed in the immunological assays of theELISA type will also be pointed out.

EXAMPLE 2 Preparation of Monoclonal Antibodies Directed Against VillinA) Immunisation Protocol

6 to 8 weeks old Balb/C mice

Antigen: purified porcine villin (10 mg/ml) (homogeneous bandcorresponding to a molecular weight of 95 Kd after electrophoresis in a10% polyacrylamide gel in the presence of SDS):

10 mM imidazole

75 mM HCl

1 mM EGTA

0.1M DTT

50% glycerol

Protocol:

Day 0: IP injection of 50 μg of pure villin in a 50/50 emulsion(PBS-complete Freund's adjuvant).

Day 14: IP booster injection of 50 μg of villin (emulsion PBS-completeFreund's adjuvant).

Day 21: IP booster injection of 50 μg of villin (emulsion PBS-completeFreund's adjuvant).

Day 28: IM injection of 20 μg of pure villin in PBS solution.

Day 29: IV injection of 10 μg of pure villin in PBS solution.

Day 32: Fusion

B) Fusion

I) Parent cells

a) Myeloma cells:

Sp2/0-Ag 14 line (8-azaguanine resistant)

Sterile culture in DMEM medium containing 10% FCS.

b) Spleen cells:

Origin: spleen of Balb/C mice hyperimmunised against porcine villin.

II) Procedure for cell fusion according to Kohler G and Milstein C(Continuous culture of fused-cells secreting antibody of predefinedspecificity, Nature 1975, 256, 495).

Fusion under sterile conditions in DMEM medium without serum, in thepresence of a 50% solution of polyethylene glycol (PEG 1,000--Merck9729) in DMEM medium without serum. After counting of the two parentalcell types, the suspension of the myeloma cells in culture is mixed withthe suspension of spleen cells in the proportion of 1 to 5.

2'30" contact.

Action of PEG stopped by dilution with complete DMEM medium.

Final dilution of the cell suspension (2×10⁵ cells/ml) in the selectiveHAT-DMEM medium.

Distribution in wells of Costar dishes--24 wells 1 ml/well (COSTARTissue Culture Cluster 24, Cat. No. 3524, COSTAR 205 Groadway,Cambridge, Mass. USA).

III) Selection of clones: Clones are identified by their capacity tosecrete antibodies directed against purified porcine villin.

C) Method of Selection

I) ELISA test: This test provides a demonstration of monoclonalanti-villin antibodies in the culture supernatants after fusion.

Fixation of antigen on a support (ELISA plaque) Concentration: 5 μg/mlin a 50 mM potassium phosphate buffer, pH 8, overnight at 4° C.

Saturation with PBS-TWEEN 20-BSA.

Incubation I with undiluted hybridoma supernatant, 3 hours at 4° C.

Incubation II with mouse anti-IgG labelled with beta -galactosidase, 2hours at 4° C.

All washings between each step are carried out with PBS-0.1% TWEEN 20.

Substrate O-nitrophenyl-beta-D-galactopyranoside (Sigma N11-27) in 0.1Mphosphate buffer, pH 7.0×10⁻³ M MgSO₄. 2×10⁻³ M MnSO₄. 2×10⁻³ Mmagnesium tritriplex, anhydrous (Merck 8409). Reading OD at 414 nm.

The clones selected are those for which the hybridoma supernatants givean OD 10 times higher than the background OD

II) Western Blotting according to Burnett, Anal. Biochem. 1981, 112,195-203: This test enables the clones secreting porcine anti-villinmonoclonal antibodies and also recognising human and chicken villin tobe selected from among the clones giving a positive ELISA test.

Various steps of the test

a) Polyacrylamide gel electrophoresis of a cell extract of chickenintestinal mucosa and of a cell extract of the line HT29 (adenocarcinomaof human colon) expressing villin.

b) Electrotransfer of the proteins separated on polyacrylamide gel tonitrocellulose paper.

c) Incubation of nitrocellulose paper to which the cell proteins hadbeen transferred with the hybridoma supernatants previously selected inthe ELISA test, overnight at 4° C.

d) Incubation for 1 hour at room temperature with mouse anti-IgGlabelled with peroxidase.

e) Substrate:

10 mg of diaminobenzidine tetrahydrochloride

20 ml 0.1M Tris HCl pH 7.6

0.2 ml of 1% H₂ O₂

All washings between each step are carried out with new-born calf serum-PBS-TRITON X100.

f) positive reaction: rapid appearance of a brown bend (molecular weight95 Kd) if an antigen-monoclonal antibody reaction has taken place.

D) Cloning of the Hybrids Selected

Method of limited dilutions:

Positive clones are diluted with selective medium so as to give only onecell per well (plate with 96 wells) containing macrophages (origin: 4week-old Balb/C mice) attached to the bottom of the well.

Addition of conditioned selective medium.

Selection of positive clones according to the methods previouslydescribed.

Recloning if necessary.

E) Preparation of Ascites

The clones selected are injected into mice (which had received aninjection of "PRISTANE" (Aldrich, 2,6,10,14-tetramethylpentadecane) 4 to5 days previously).

1 to 2×10⁶ cells injected/mouse.

Recovery, 10 to 15 days later, of the ascitic fluid containing theclonal cells which have proliferated. The concentration of anti-villinmonoclonal antibody in the ascitic fluid is higher than 1 mg/ml.

Freezing of the selected clones in DMEN medium containing 10% FCS-5%DMSO.

Storage in liquid nitrogen at -176° C.

Direct ELISA Procedure for Titration of Villin

Adsorption of purified IgGs from the ascite BD-ID2C3 at constantconcentration on ELISA plaques.

The antibody was purified by ion exchange chromatography (DEAE-Trisacryl) and on a hydroxyapatite column.

Concentration to be determined.

Incubation for 2 hours at 37° C., then overnight at 4° C.

Washing with PBS/0.1% TWEEN 20.

Saturation of the plaques: Incubation for 30 minutes in the presence ofPBS/0.1% TWEEN 20/0.4% BSA.

Addition of antigen

Serial dilutions of purified villin of decreasing concentration from 5μg/ml to 1 or 0.1 ng/ml in the presence of 0.4% BSA.

Incubation for 3 hours at 4° C.

Washings with PBS/0.1% TWEEN 20.

Addition of purified IgG obtained from a polyclonal rabbit serumdirected against villin and coupled to beta-galactosidase.

Addition of 20 mg of p-nitrophenyl-beta-D-galactopyrannoside from asolution containing 4 mg/ml to 20 ml of 0.1M phosphate buffer, pH 7,10⁻³ M MgSO₄, 2×10⁻⁴ M MnSO₄, 2×10⁻³ M EDTA (Merck 8409)

Incubation for x hours at 37° C.

Optical density reading at 414 nm.

EXAMPLE 3 Clinical Observations

Measurements have been made of the amounts of villin detectable in theserum by making use of the ELISA test described below, the sensitivityof which makes it possible to detect very low levels of villin in a cellextract (0.5 ng of villin per mg of total protein). In fact, theinventors have confirmed that necrosis of cells containing villin, anintracellular protein expressed by normal and tumor cells of thedigestive tract, may lead to its liberation into the circulation undercertain physiopathological conditions.

A study conducted on a population of blood donors (n=190) showed thatvillin was not detectable in the vast majority (n=168) of the sera.However, a detectable concentration of villin was present in the serumof a small number of individuals (n=15) (5 to 10 ng/ml). This value isregarded as a concentration of villin which has no pathologicalsignificance. There were also several individuals (n=7) in whom thelevel of villin was indeed higher than the basal value (50 to 100ng/ml). The study of these so-called "normal" individuals (3.6%) was notextended to include a clinical examination of their digestive tract(fiber optic endoscopy, colonoscopy).

The initial results obtained in different gastro-intestinal diseases aresummarized in the table below. Detection of measureable amounts (valueslying between 10 and 10,000 ng/ml) of villin in the sera of patientssuffering from:

Malignant diseases of the digestive tract:

colorectal cancers

gastric cancers

Benign diseases of the digestive tract:

villous tumors

Crohn's disease

ulcerative colitis

ulcers of the duodenal, gastric and esophagal bulb.

On the other hand, it is important to note that polyadenomas of thedigestive tract ("polyps") do not lead to the liberation of this proteininto the circulation.

This study also enabled certain other deductions to be made:

1. There seems to be no correlation between the amount of circulatingvillin and the size and stage of development of the tumor.

2. Measurement of this protein during the post-operative follow-upshowed a fall in its serum concentration after complete surgicalexcision. On the other hand, in cases in which tumor tissue persisted(incomplete excision, recurrences, metastases), the level of villin wasobserved to remain stationary or increase.

This study indicated that villin is not usually detectable in the serumof normal subjects, whereas it can be measured in the serum of patientswho have contracted malignant diseases (colic or rectal cancers) or whoare suffering from benign ulcerations (diseases such as Crohn's disease,ulcerative colitis, ulcers) of the digestive tract which provoke theintervention of necrotic and inflammatory processes of the digestivemucous.

The measurement of the concentration of villin in the blood also provedto be of importance in the surveillance of benign diseases (ulcerationsand inflammatory diseases of the digestive tract). Moreover, in thestudy of circulating villin, it is possible to use the antibodies of theinvention in conjunction with other tumoral markers such as CEA(carcinoembryonic antigen), which is presently used for the surveillanceof the evolution of cancers but the principal disadvantages of whichare: 1. low specificity for the organ affected; 2. its presence invarious benign diseases.

EXAMPLE 4 Preparation of cDNA Corresponding to Human Villin Preparationof the mRNAs

The mRNAs are prepared from the HT29 cell line derived from a humancolon adenocarcinoma. The RNAs are extracted by the guanidium chloridemethod described by Ullrich et al. in Science, 1977, 196, 1313-1319. Thepurification of the poly A⁺ mRNAs is carried out by chromatography on acolumn of oligo dT-cellulose according to the method described by Avivand Leder in Proc. Natl. Acad. Sci. USA, 1972, 69, 1408-1412.

Preparation of cDNAs

The first and second strand of the cDNAs are prepared according to themethod of Fiddes and Goodman described in Nature, 1979, 291, 351-355.the sequential addition of the linkers SalI at the 3' end and Bam H1 atthe 5' end is carried out by the method of Helfmann et al. reported inProc. Natl. Acad. Sci. USA, 1983, 80, 31-35.

This leads to a cDNA bank containing about 30,000 recombinant clones.

Vector

Plasmids pEX 1-3 (see Stanley and Luzio, EMBO Journal, 1984, 3,1429-1430). These vectors derive from plasmids containing the cro-lacZgene fusion, the expression of which is under the control of thepromoter PR of the bacteriophage lambda. A polynucleotide containingseveral unique restriction sites was inserted at the 3' end of the lacZgene. The termination signals for transcription and translation are alsoinserted in the three reading frames.

The cDNAs were inserted in each of the pEX plasmids under the directionof the restriction enzymes Bam H1 and SalI. Thus, they all have the sameorientation with respect to the cro-lacZ gene.

Transformation

The bacterial strain used is E. coli pop 2135, constructed by O. Raibaudas described in Nucleic Acid Research in the following manner:

The 2.3 kb fragment BglII of the phage lambda, carrying the C1857 alleleand the promoter P_(R) is cloned into the Bam H1 site of a polylinker asindicated in the following scheme: ##STR3##

The EcoRI fragment thus obtained is then cloned into the EcoRI site ofpOM41 and transferred into the chromosome of the strain C600 (see Gene,29, 231-241).

By cotransduction using P1' with a marker linked to aroB, this structureis introduced into the background MM294 (F₃₁ ⁻ endA thi hsdR). E. colipop 2135 is obtained: orientation maIT, C1857, P_(R), maIPO.

The transformation of the bacterial strain E. coli pop 2135 by therecombinant plasmids is carried out according to the technique usingrubidium described by Hanahan et al., J. Mol. Biol., 1983, 166, 557-580.This strain contains the allele cIts857 of the cro repressor.

The number of recombinants obtained is of the order of 10³ to 10 ⁴ ng ofcDNA.

Selection of Clones by Immunological Detection

The recombinant clones obtained are spread on nitrocellulose filters.The synthesis of the hybrid protein is induced during 2 hours ofincubation at 42° C. After lysis of the bacteria by SDS and renaturationof the proteins in the absence of methanol, the clones are analysed byimmunological screening. Renaturation of the proteins is carried outaccording to the technique of Burnett reported in Anal. Biochem. 1981,112, 195-203.

In the first step, the bank is screened by a polyclonal antibodydirected against intact porcine villin. A secondary screening is thencarried out using a polyclonal antibody directed against a COOH-terminalfragment of chicken villin and two monoclonal antibodies (BD-ID2C3 andIID₃ H₉) recognizing epitopes located in the COOH-terminal region of themolecule.

The clone pEXZ-V19 contains an insert of 510 base pairs. The codingregion represents 310 base pairs. It is followed by non coding sequencesof 200 base pairs.

The cloned cDNA codes for the 95 COOH-terminal amino acids of humanvillin and represents about 1/10 of the total protein (molecular weight:95 Kd).

REFERENCES

(1) Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E.,Rutter, W. J. & Goodman, H. M. Rat insulin genes: construction ofplasmids containing the coding sequences. Science, 1977, 196, 1313-1319.

(2) Aviv, H. & Leder, P. Purification of biologically active globin mRNAby chromatography on oligothymidylic acid cellulose. Proc. Natl. Acad.Sci. USA, 1972, 69, 1408-1412.

(3) Fiddes, J. C. & Goodman, H. M. Isolation, cloning and sequenceanalysis of the cDNA for the alpha-subunit of human chorionicgonadotropin. Nature, 1979, 281, 351-355.

(4) Helfman, D. M., Feramisco, J. R., Fiddes, J. C., Thomas, G. P. &Hughes, S. H. Identification of clones that encode chicken tropomyosinby direct immunological screening of a cDNA expression library. Proc.Natl. Acad. Sci. USA, 1983, 80, 31-35.

(5) Stanley, K. K. & Suzio, J. P. Construction of a new family of highefficiency bacterial expression vectors: identification of cDNA clonescoding for human liver. EMBO Journal, 1984, 3, 1429-1434.

(6) Stanley K. K. Solubilization and immune-detection ofbeta-galactosidase hybrid proteins carrying foreign antigenicdeterminants. Nucleic Acids Res., 1983, 11, 4077-4092.

(7) Hanahan, D. Studies on transformation of E. coli with plasmids. J.Mol. Biol., 1983, 166, 557-580.

(8) Burnett, W. N. "Western Blotting": Electrophoretic transfer ofproteins from sodium dodecyl sulfate-polyacrylamide gels to unmodifiednitrocellulose and radiographic detection with antibody andradioiodinated protein A. Anal. Biochem., 1981, 112, 195-203.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 528 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAGTGGAGTAACACCAAATCCTATGAGGACCTGAAGGCGGAGTCTGGCAACTCTAGGGAC60                TGGAGCCAGATCACTGCTGAGGTCACAAGCCCCAAAGTGGACGTGTTCAATGCTAACAGC120               AACCTCAGTTCTGGGCCTCTGCCCATCTTCCCCCTGGAGCAGCTAGTGAACAAGCCTGTA180               GAGGAGCTCCCCGAGGGTGTGGACCCCAGCAGGAAGGAGGAACACCTGTCCATTGAAGAT240               TTCACTCAGGCCTTTGGGATGACTCCAGCTGCCTTCTCTGCTCTGCCTCGATGGAAGCAA300               CAAAACCTCAAGAAAGAAAAAGGACTATTTTGAGAAGAGTAGCTGTGGTTGTAAAGCAGT360               ACCCTACCCTGATTGTAGGGTCTCATTTTCTCACCGATATTAGTCCTACACCAATTGAAG420               TGAAATTTTGCAGATGTGCCTATGAGCACAAACTTCTGTGGCAAATGCCAGTTTTGTTTA480               ATAAATGTACCTATTCCTTCAGAAAGATGATACCCCAAAAAAAAAAAA528                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LysTrpSerAsnThrLysSerTyrGluAspLeuLysAlaGluSerGly                              151015                                                                        AsnSerArgAspTrpSerGlnIleThrAlaGluValThrSerProLys                              202530                                                                        ValAspValPheAsnAlaAsnSerAsnLeuSerSerGlyProLeuPro                              354045                                                                        IlePheProLeuGluGlnLeuValAsnLysProValGluGluLeuPro                              505560                                                                        GluGlyValAspProSerArgLysGluGluHisLeuSerIleGluAsp                              65707580                                                                      PheThrGlnAlaPheGlyMetThrProAlaAlaPheSerAlaLeuPro                              859095                                                                        ArgTrpLysGlnGlnAsnLeuLysLysGluLysGlyLeuPhe                                    100105110                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTATC60                GAATTC66                                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ValPheThrAlaThrThrThrLeuValProThrLysLeuGluThrPhe                              151015                                                                        ProLeuAspValLeuValAsnThrAlaAlaGluAspLeuProArgGly                              202530                                                                        ValAspProSerArgLysGluAsnHisLeuSerAspGluAspPheLys                              354045                                                                        AlaValPheGlyMetThrArgSerAlaAsnLeuProLeuTrpLysGln                              505560                                                                        GlnAsnLeuLysLysGluGluLysGlyLeuPhe                                             657075                                                                        __________________________________________________________________________

We claim:
 1. A mouse monoclonal antibody which binds human villin, porcine villin, chicken villin, and rat villin.
 2. A hybridoma that produces a mouse monoclonal antibody which binds human villin, porcine villin, chicken villin, and rat villin.
 3. A mouse monoclonal antibody which binds human villin, porcine villin, chicken villin, and rat villin, wherein said monoclonal antibody has a detectable label. 